Automated enzymatic assay for plasma ammonia

Abstract

An enzymatic method for determining plasma ammonia with the Du Pont Automatic Clinical Analyzer (aca) is described. The assay requires a sample volume of 500 μL for a kinetic ammonia measurement. The reaction is initiated with glutamate dehydrogenase and the rate of depletion of NADPH is monitored with two measurements, 17 s apart, at 340 nm. Reaction conditions have been optimized for maximum sensitivity through both one-factor-at-a-time and multiple variable response surface optimization techniques. Linearity to 1000 μmol of ammonia per liter of plasma has been achieved. No significant interferences were observed from anticoagulants or endogenous blood components, including pyruvate and oxalacetate. Use of the coenzyme NADPH (instead of NADH) in this aca procedure eliminates the lengthy pre-incubation otherwise required for endogenous dehydrogenase reactions.